Project Summary Abstract During the cell cycle, regulatory mechanisms are in place to ensure that the genome is copied and properly inherited by daughter cells. In contrast, little is known about whether regulatory mechanisms are present to ensure inheritance of cytoplasmic organelles. A vital organelle, the endoplasmic reticulum (ER) produces virtually all secretory proteins, transmembrane proteins, and proteins of the secretory pathway organelles. The ER is also the birthplace of cellular lipids. Proper inheritance of the ER is thus critical for the cell. We have now identified a cell cycle regulatory mechanism, the ER Stress Surveillance pathway or ERSU, that ensures the proper inheritance of a functional ER during the cell cycle. ERSU is distinct from any other previously described signaling pathway including the well-known UPR pathway. When ERSU is activated by ER stress, compromised ER can still enter the daughter cell, but cannot anchor at the bud tip and so retracts. We find that ERSU is a novel cell cycle checkpoint that then halts the cell cycle until functional ER is available. In our initial analysis, we have identified critical ERSU pathway components including WSC1, a cell surface signaling protein, and SLT2, a MAP kinase. Deletion of either gene eliminates ERSU: bad ER is now anchored and inherited, but renders the daughter bud non-viable. We will investigate the ERSU pathway as follows: In AIM I, we will define the ER initiator(s) of ERSU. We have strong preliminary evidence that lipid synthesis enzymes are key to initiation. Interestingly, lipids are known to play a role in many mammalian heath-related signaling pathways including asthma, as we have recently reported. In AIM 2, we will dissect the mechanism by which ER inheritance is blocked in response to ER stress. We will use a recently developed live-cell assay that allows us to view ER inheritance while it is under stress. ER entry, anchoring, and the fate of the daughter will be examined both in wild type cells and in our increasing number of ERSU pathway- defective mutants. We also will drill down at the molecular level by examining specific ER-anchoring components for ERSU-induced alteration. In Aim 3, we will for the first time probe the cell cycle boundaries of ERSU and, indeed, of the UPR. We will study the relationship between cell cycle stages and ERSU. For example, can ERSU be induced at any phase of the cell cycle? A failure to regulate ER functional capacity is increasingly recognized as a contributing factor to the pathophysiology of many human diseases, including certain cancers. Thus, knowledge of the cellular mechanism that assures inheritance of a functionally competent ER will be invaluable towards the development of previously unrecognized strategies for therapeutic intervention.